摘要
用限制酶EcoRⅠ、KpnⅠ分别对雅致枝霉As3.2806基因组DNA进行消化,而后在低浓度条件下利用T4DNA连接酶使DNA自身环化。根据已知基因序列,设计一对长度为35nt的长反向引物和两对较短的引物,以基因组连接产物为模板,通过三轮嵌套式PCR反应,获得一长度约为4kb的扩增片段。经序列测定表明得到了Δ6-脂肪酸脱氢酶基因上游序列约为1.3kb,初步序列分析显示该序列为一潜在的启动子序列。
Thamnidium elegans is a kind of phycomycete that produces essential unsaturated fatty acids, particularly γ-linolenic acid. In this process, Δ^6-Fatty acid desaturase （D6D） plays a key role due to its enzymatic properties that catalyze the Δ^6 site dehydrogenation of precursor linoleic acid （ 18 ： 2Δ^9, 12 n-6） and a-linolenic acid （ 18 ： 3Δ9. 12,15 n-3 ）. This reaction is the first and rate-limiting step of highly unsaturated fatty acids（HUFA） synthesis pathways. After we have isolated and cloned the gene coding Δ^6-fatty acid desaturase from Thamnidium elegans As3.2806 （GenBank accession number DQ099380）, our interest focuses on the promotion and regulation of the gene transcription. To achieve this aim, we designed long primers and used nested inverse PCR to amplify DNA flanking sequences. First, genome of Thamnidium elegans was extracted and digested with restriction enzymes EcoR Ⅰ and Kpn Ⅰ , respectively. Then we ligated the digested DNA with T4 ligase at low concentration which is propitious for linear DNA to joint intromolecule. According to the sequence of Δ^6-fatty acid desaturase gene of Thamnidium elegans, we designed a couple of 35nt long inverse primers and two couples of shorter inverse primers for inverse PCR. Three rounds of PCR reactions were performed. In the primary reaction, the ligated DNA was used as a template, and the product was used as the template of the secondary reaction, the tertiary reaction was achieved in the same way. After all the three rounds of reactions, we got a nice product about 4kb from the EcoR I digested sample, in which a 1.3kb 5＇ upstream sequence （GenBank accession number DQ309425） of △^6-fatty acid desaturase gene containing several putative regulatory elements including TATA box, FSE-2, AP-1 sites, CCAAT cis-element site and STRE-binding site was derived after sequencing. All of these implied intensely that this 1.3kb fragment is a condition-regulated promoter. It is the first report about Thamnidium elegans △^6-fatty acid desaturase gene promoter. The procedure described here is a rapid and simple method and particularly useful to isolate flanking sequences from fungal genome.
出处
《生物工程学报》
CAS
CSCD
北大核心
2006年第4期581-586,共6页
Chinese Journal of Biotechnology
基金
国家自然科学基金资助项目(No.30200176)
天津市自然科学基金资助项目(No.013802511)。~~
